The BOLD-fMRI approach has revolutionized assessment of brain function. This complex effect represents MRI signal changes that are consequential to a hemodynamic response secondary to activation. As the BOLD response reflects the changing oxygen extraction fraction, OEF, it allows some insight into basic brain physiology. However, such interpretations are confounded by multiple contributions to the effect. BOLD is sensitive not only to intravascular oxygenation changes, but also to extravascular effects of such changes around parenchyma and around veins draining from activated cortex. In addition, BOLD is influenced by alterations in voxel composition due to changes in cerebral blood volume (CBV). Contributions of these effects to the BOLD signal intensity changes depend on magnetic field strength, voxel size, type of MRI experiment (spin echo/gradient echo). Thus, there are many variables and few observable parameters. In the previous period we have developed a new fMRI approach that can add several essential observables, namely tissue fractions, CBV and the tissue relaxation rates. We also re-interpreted the BOLD post-stimulus undershoot in terms of continued oxygen metabolism and showed that, surprisingly, BOLD spin-echo signals can occur in draining veins. The ultimate goal of this proposal is to gain a complete quantitative understanding of the physiological, physical, and spatial aspects of the BOLD effect. In AIM 1 we propose experiments to confirm the proposed oxygen-metabolism based character of the BOLD post-stimulus undershoot and to develop strategies to use this undershoot for fMRI voxel selection. In AIM 2 we propose experiments that can distinguish intravascular, extravascular, macrovascular, and microvascular BOLD components. These data will be used to evaluate existing extravascular BOLD theories. In AIM 3 we measure the blood transverse relaxation rates for blood in which the transport properties of erythrocyte water channels are impaired by an aquaporin channel blocker. This will be combined with our previous data without blocker to evaluate the effect of exchange versus magnetic field gradients on the intravascular BOLD effect. Based on our blood data acquired in the previous grant period and our in vivo data proposed to be acquired here, we will derive a quantitative description for the BOLD effect that has only physiological and physical parameters. This formalism should allow description of the BOLD effect for all physiological perturbations.